The Ehrlichiae are obligate intracellular bacteria found in circulating leukocytes of infected animals. Ehrlichia canis (E. canis) infects monocytes and causes ehrlichiosis in animals belonging to the family Canidae. E. canis is transmitted by the brown dog tick, Rhipicephalus sanguineus. 
Canine monocytic ehrlichiosis (CME) consists of an acute and a chronic phase. The acute phase is characterized by fever, serous nasal and ocular discharges, anorexia, depression, and loss of weight. The chronic phase is characterized by severe pancytopenia, epistaxis, hematuria, blood in feces in addition to more severe clinical signs of the acute disease. If treated early during the course of the disease, dogs respond well to doxycycline. However, chronically infected dogs do not respond well to the antibiotic. Therefore, early diagnosis is very important for treating canine ehrlichiosis.
Human monocytic ehrlichiosis (HME) is a tick-borne, emerging infectious disease that is caused by the rickettsial pathogen, Ehrlichia chaffeensis (E. chaffeensis). The course of HME begins with an asymptomatic pre-patent period, followed by an acute phase where the vertebrate host suffers pyrexia, anorexia, weight loss, cytopenia and even death. Non-human hosts that survive the acute phase typically undergo partial recovery and suffer mild chronic infections, during which they could be persistent carriers that are capable of infecting tick vectors. Both dogs and white tailed deer are susceptible to infection with E. chaffeensis, and both of these hosts are suspected reservoirs of this pathogen. E. chaffeensis appears to be transmitted by Amblyomma. americanum, and appears to be endemic to the southern U.S. where this tick is indigenous. Symptoms of human monocytic ehrlichiosis (HME) are similar to those of canine monocytic ehrlichiosis (CME) that is also known as tropical canine pancytopenia. (Hildebrandt, P. K., D. L. Huxsoll, et al. (1973 (1973). Pathology of canine ehrlichiosis (tropical canine pancytopenia). Am J Vet Res 34(10): 1309-20.; Kuehn, N. F. and S. D. Gaunt (1985). Clinical and hematologic findings in canine ehrlichiosis. J Am Vet Med Assoc 186(4): 355-8.; Eng, T. R., J. R. Harkess, et al. (1990). Epidemiologic, clinical, and laboratory findings of human ehrlichiosis in the United States, 1988. Jama 264(17): 2251-8.; McDade, J. E. (1990). Ehrlichiosisxe2x80x94a disease of animals and humans. J Infect Dis 161(4): 609-17.) The etiologic agents of CME and HME, E. canis and E. chaffeensis, respectively, have been placed in the same genogroup based on 16S rRNA sequences and antigenic cross-reactivity Anderson, B. E., J. E. Dawson, et al. (1991). Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol 29(12): 2838-42.
The primary test for diagnosing CME or HME is the indirect fluorescent antibody (IFA) test. This test uses the etiologic agents Ehrlichia canis or E. chaffeensis, respectively, to diagnose infection. The IFA test, however, has serious limitations. The IFA test is subject to false positives because the antigens are whole infected cells which comprise many nonspecific proteins that can cross-react with sera from some patients. The IFA test is also subject to false negatives because IFA antigens are unstable and may become inactivated during storage. In addition the IFA test requires a special equipment to perform the test. For example, the IFA test requires a tissue culture system for growing the bacterium that are used to prepare the antigen slides, a fluorescent microscope, and trained persons to evaluate the serum reactivity to the bacterial antigen on the slide.
Serodiagnosis is another method which has been developed to diagnose canine or human ehrlichiosis. The method involves testing the blood of the animal for antibodies immunoreactive with outer membrane proteins of these pathogens. Serodiagnosis cannot be used until the infected subject has produced such antibodies. Accordingly, serodianosis cannot be used early during the course of infection. Moreover, serodiagnosis cannot be used for detecting an ongoing infection.
Accordingly, it is desirable to have additional methods and tools which can be used for diagnosing canine and human ehrlichiosis, particularly methods and tools which can be used to detect an ongoing infection. Methods and tools which can be used to detect E. canis and E. chaffeensis in the invertebrate vectors which transmit these pathogens to their respective vertebrate hosts are also desirable.
The present invention provides tools and methods for detecting the presence of E. canis and E. chaffeensis in a sample obtained from an animal, particularly from a member of the Canidae family. The method for detecting E. canis comprises providing a p30 primer set comprising a first primer having a sequence which is complementary to a sequence on the E. canis p30 gene sense strand and a second primer which is complementary to a sequence which is complementary to a sequence on the E. canis p30 gene of antisense strand, amplifying DNA in the sample using a polymerase chain reaction and the p30 primer set, and determining the length or sequence of the PCR products, wherein the presence of a PCR product having a sequence or length which corresponds to the sequence or length of the region of the p30 gene which is located between the nucleotide sequences to which the first p30 primer and the second p30 primer bind is indicative of the presence of E. canis in the sample.
The present invention also relates to the p30 primer sets. Each p30 primer set comprises a first p30 primer and the second p30 primer, both of which are from 15 to 35 nucleotides in length. The first p30 primer, i.e., the forward primer, comprises a sequence which is complementary to a consecutive sequence, preferably of at least 14 nucleotides in length, within the following sequence: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30 primer, i.e. the reverse primer, comprises a sequence which is complementary to the inverse complement of a consecutive sequence, preferably of at least 14 nucleotides in length, contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2. Such primers are useful for detecting the presence of E. canis in members of the Canidae family. Such primers are also useful for detecting the presence of E.canis DNA in samples obtained from ticks or other invertebrate carriers which feed on the vertebrate hosts.
The method for detecting E. chaffeensis comprises providing a p28 primer set comprising a first primer comprising a sequence which is complementary to a sequence on the E. chaffeensis p28 gene sense strand and a second primer which is complementary to a sequence on the E. chaffeensis p28 gene antisense strand, amplifying DNA in the sample using a polymerase chain reaction and the p28 primer set, and determining the length or sequence of the PCR products, wherein the presence of a PCR product having a sequence or length which corresponds to the sequence or length of that portion of the p28 gene which is located between the sequence to which the first p28 primer and second p28 primer bind is indicative of the presence of E. chaffeensis DNA in the sample.
The present invention also relates to the primers in the p28 primer set. The first p28 and second p28 primers are from 15 to 35 nucleotides in length. The first p28 primer, i.e. the forward primer, comprises a sequence which is substantially identical to the complement of a consecutive sequence, preferably of at least 14 nucleotides in length, within the following sequence :A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO.3.
The second primer, i.e. the reverse primer, comprises a sequence which is complementary to the inverse complement of a consecutive sequence, preferably of at least 14 nucleotides in length, within one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO.4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO.5. Such primers are useful for detecting the presence of E. chaffeensis in samples obtained from vertebrate animals such as humans or dogs, or from the invertebrate vectors which transmit this pathogen from one vertebrate animal to another.